protein domain array membranes containing sh2, sh3, pdz, or ww domains  (Panomics Inc)

Journal: Journal of Cell Science

Article Title: Src binds cortactin through an SH2 domain cystine-mediated linkage

doi: 10.1242/jcs.121046

Figure Lengend Snippet: Src SH2 binding to cortactin does not involve tyrosine phosphorylation and binds cortactin repeats 1 and 5. (A) GST and GST-Src SH2 affinity precipitation from MTLn3 cells evaluated for cortactin tyrosine phosphorylation. The ratio of phosphorylated cortactin levels and the normalized amounts of total precipitated cortactin are indicated. (B) Src SH2 far western analysis of FLAG-tagged recombinant wild-type and cortactin phosphorylation mutants. TYM, triple tyrosine mutant. (C) Affinity precipitation analysis of FLAG-tagged recombinant wild-type and cortactin phosphorylation mutants from extracts with GST-Src SH2 domain. (D) Affinity precipitation of non-phosphorylated, recombinant cortactin with GST and GST-Src SH2. Normalized intensity levels are shown relative to GST control. (E) GST-SH2 domain far western blotting of the cortactin NTA and repeats region. CT, C terminus; LC, IgG light chain; NT, N terminus. Arrows denote position of IgG heavy chain (HC) recognized by cross reactivity with secondary antibodies during the blotting process. Asterisks indicate the positions of recombinant cortactin proteins. (F) GST-SH2 far western analysis of cortactin deletion cortactin constructs. (G) Far western binding of the GST-Src SH2 domain to tandem cortactin repeat chimeric constructs.

Article Snippet: Screening of TransignalTM SH2 Domain Arrays (Panomics Cat. NO. MA3040) was conducted using 1.0 mg of each cortactin peptide according to the manufacturer's protocol.

Techniques: Binding Assay, Phospho-proteomics, Affinity Precipitation, Western Blot, Recombinant, Mutagenesis, Control, Far Western Blot, Construct

Journal: Journal of Cell Science

Article Title: Src binds cortactin through an SH2 domain cystine-mediated linkage

doi: 10.1242/jcs.121046

Figure Lengend Snippet: Cortactin cysteines 112 and 246 are required for Src SH2 domain binding. (A) Alignment of cortactin repeats denoting C112 and C246. (B,C) Far western blotting of GST-Src SH2 domain with cortactin cysteine to alanine mutants. CT, C terminus; HC, IgG heavy chain; LC, IgG light chain; NT, N terminus; WT; full-length wild-type cortactin. Dashed line separates HC from chimeric cortactin proteins (asterisks) due to similar molecular weights. (D) Far western analysis of Src SH2 domain binding to the C112/C246A cortactin double cysteine mutant (DCM). (E) Affinity precipitation analysis of Src SH2 domain binding to the C112/C246A cortactin double cysteine mutant (DCM).

Article Snippet: Screening of TransignalTM SH2 Domain Arrays (Panomics Cat. NO. MA3040) was conducted using 1.0 mg of each cortactin peptide according to the manufacturer's protocol.

Techniques: Binding Assay, Far Western Blot, Western Blot, Mutagenesis, Affinity Precipitation

Journal: Journal of Cell Science

Article Title: Src binds cortactin through an SH2 domain cystine-mediated linkage

doi: 10.1242/jcs.121046

Figure Lengend Snippet: Cysteine-containing cortactin peptides dock within the Src SH2 phosphotyrosine binding region. (A) Molecular modelling of the Src SH2 domain with phosphorylated Src and cortactin pentapeptides. Enlarged views show position of Src R175, Src C185 and the respective central Src or cortactin peptide residues. (B) Calculated binding energies for each peptide docking condition shown in (A).

Article Snippet: Screening of TransignalTM SH2 Domain Arrays (Panomics Cat. NO. MA3040) was conducted using 1.0 mg of each cortactin peptide according to the manufacturer's protocol.

Techniques: Binding Assay

Journal: Journal of Cell Science

Article Title: Src binds cortactin through an SH2 domain cystine-mediated linkage

doi: 10.1242/jcs.121046

Figure Lengend Snippet: Binding and phosphorylation of cortactin by Src is redox dependent and requires Src C185. (A) Co-immunoprecipitation (IP) of cortactin with Src followed by analysis under reducing (R) and non-reducing (NR) conditions. Asterisks denote equivalent bands in cortactin and Src immunoblots. (B) Co-immunoprecipitation of Src with cortactin followed by analysis under reducing (R) and non-reducing (NR) conditions. Asterisks denote equivalent bands in cortactin and Src immunoblots. (C) Phosphorylation of cortactin by Src in the absence and presence of DTT. (D) Far western analysis of GST-Src SH2 and C185A. (E) Affinity precipitation analysis of FAK and cortactin binding to GST-Src SH2 C185A.

Article Snippet: Screening of TransignalTM SH2 Domain Arrays (Panomics Cat. NO. MA3040) was conducted using 1.0 mg of each cortactin peptide according to the manufacturer's protocol.

Techniques: Binding Assay, Phospho-proteomics, Immunoprecipitation, Western Blot, Affinity Precipitation

Journal: Journal of Cell Science

Article Title: Src binds cortactin through an SH2 domain cystine-mediated linkage

doi: 10.1242/jcs.121046

Figure Lengend Snippet: Src C185 forms a cystine bond with cortactin C112 and C246. (A) Sequence of the predicted Src SH2 domain tryptic fragment containing C185. Predicted cystine bonding between Src C185 and the cortactin C112 and C246 tryptic peptides with predicted masses are shown below. (B–D) Extracted ion chromatogram (left) and ion fragmentation spectra (right) from tandem LC-MS/MS of the GST-Src SH2 domain (B), the GST-SH2 domain with the cortactin C112 peptide (C) and the GST-SH2 domain with cortactin C246 peptide (D). Spectra were enlarged to indicate the position of the Src C185 b4 ion (boxed in red).

Article Snippet: Screening of TransignalTM SH2 Domain Arrays (Panomics Cat. NO. MA3040) was conducted using 1.0 mg of each cortactin peptide according to the manufacturer's protocol.

Techniques: Sequencing, Liquid Chromatography with Mass Spectroscopy

Journal: Journal of Cell Science

Article Title: Src binds cortactin through an SH2 domain cystine-mediated linkage

doi: 10.1242/jcs.121046

Figure Lengend Snippet: Observed and predicted ion masses of GST-Src-SH2, GST-Src-SH2 + cortactin C112 and GST-Src-SH2 + cortactin C246 tryptic peptides

Article Snippet: Screening of TransignalTM SH2 Domain Arrays (Panomics Cat. NO. MA3040) was conducted using 1.0 mg of each cortactin peptide according to the manufacturer's protocol.

Techniques:

Journal: Journal of Cell Science

Article Title: Src binds cortactin through an SH2 domain cystine-mediated linkage

doi: 10.1242/jcs.121046

Figure Lengend Snippet: Model of cysteine-mediated interactions in cortactin regulation. (A) Model of cysteine-based cortactin activation and phosphorylation by Src. (B) Phylogenetic co-conservation of cortactin C112/246 and Src C185. Conserved cysteines are in red and the homologous positions highlighted in yellow. (C) Cartoon representation of Src SH2 binding to phosphotyrosine and cysteine residues. Src amino acids 172–191 within the SH2 domain binding pocket are in white; interacting arginine 175 and cysteine 185 residues are in red. Cystine bonding is indicated as a red line. Phosphotyrosine and cystine binding ligands are listed. (D) Alignment of cysteine-containing SH2 domains. Domains known to bind ligands in a phosphotyrosine-independent manner are in bold italics. Cysteine residues are in red. Shading: green; hydrophobic, blue; positively charged, red; negatively charged, yellow; polar.

Article Snippet: Screening of TransignalTM SH2 Domain Arrays (Panomics Cat. NO. MA3040) was conducted using 1.0 mg of each cortactin peptide according to the manufacturer's protocol.

Techniques: Activation Assay, Phospho-proteomics, Binding Assay